Bacillus anthracis is surrounded by an antiphagocytic capsule that is composed of poly-?-D-glutamic acid (? DPGA). The ? DPGA capsule is an attractive target for immunoprotection because it is essential for virulence and offers a second target for vaccine development in addition to the currently targeted protective antigen. Indeed, there is considerable potential for synergy in a dual target strategy. However, targeting the B. anthracis capsule for active or passive immunization will require an understanding of how capsular antibodies mediate protection and an identification of in vivo correlates of protection. Such information is particularly important with a select agent for which there is little naturally occurring disease that would allow for evaluation of active or passive immunization efficacy. Six monoclonal antibodies (mAbs) reactive with ? DPGA were generated and studied during the current grant period. Most were of the IgG3 subclass. Five were protective;one was not. IgG subclass switch families (IgG3 ->IgG1 ->IgG2b ->IgG2a) were generated from two of the protective mAbs. Despite having identical variable regions, the IgG1, IgG2b and IgG2a variants failed to protect. Features common to all protective mAbs were i) the IgG3 subclass, ii) high intrinsic affinity, and iii) and the ability of the antibody to structurally remodel the capsular outer edge. The overall goal of this competing renewal is to understand, at a molecular level, why one ? DPGA mAb is protective and another ? DPGA mAb fails to protect. The overall hypothesis is that the IgG subclass- dependent ability of an antibody to protect is dependent on i) the ability of the mAb to remodel the capsular edge in an affinity-dependent manner, ii) the inherent biological activities of each subclass, and iii) the response of effector cells to signaling via activation or inhibitory Fc receptors. This hypothesis will be examined in five Specific Aims that will examine the roles of IgG subclass and affinity as independent variables that influence protection. Specific Aim 1 will identify the IgG heavy chain domains that influence mAb affinity. Specific Aim 2 will assess the roles of IgG subclass and affinity in protection. Specific Aim 3 will evaluate synergy between mAbs of different IgG subclasses. Aim 4 will examine the role of FcR signaling in protection. Finally, Aim 5 will utilize information generated in Aims 1-4 to optimize parameters for prevention and treatment of murine and rabbit pulmonary anthrax. PUBLIC HEALTH RELEVANCE: This project will determine the mechanism for protection by antibodies directed toward the outer coat of Bacillus anthracis. This information will be critical to decisions made in formulation of a new-generation anthrax vaccine or the design of genetically engineered antibodies to treat anthrax in humans.